Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Fathi Halaweish


Cucurbitacins, Cytotoxicity, Epidermal growth factor receptor, Estrone analogs, Non-small cell lung cancer, Triple Negative Breast Cancer


Cancer is second only to cardiovascular illnesses as the deadliest human disease globally. Currently, non-small cell lung cancer (NSCLC) and triple negative breast carcinoma (TNBC) are the most frequent types of cancer and have the highest mortalities. Epidermal growth factor receptor (EGFR), a central regulator of tumor progression, is frequently overexpressed in both cancers and is a key clinical target for therapeutic intervention. Natural products and their synthetic analogs have been utilized as EGFR tyrosine kinase inhibitors (TKIs) with potent antitumor effects. However, acquired resistance limits the long-term efficacy of these drugs. Estrone has been used as a scaffold in some studies where pharmacophores with antitumor properties were introduced to generate lead candidates with improved efficacy and safety. In our research group, estrone is employed as a starting material to synthesize novel hybrid analogs. Many of our estrone analogs, especially those bearing cucurbitacin pharmacophores have been documented to exhibit potent cytotoxic effects against distinct cancers. However, these hybrid analogs have never been explored as potential agents targeting EGFR-dependent cancers. Here, we describe the cytotoxic effects of novel estrone analogs: a hybrid of estrone base-scaffold and modified cucurbitacin (MMA series) or triazole (Fz series) pharmacophores as potent agents against EGFR-dependent cancers, viz. NSCLC (NCIH226) and TNBC (MDA-MB- 231, MDA-MB-468) tumor models. Molecular docking studies were carried out with OpenEye software. The MTT cell viability and trypan blue stain assays were used to perform cytotoxicity studies. Morphological changes and cell cycle arrest were carried out by microscopy and flow cytometric techniques, respectively. Annexin V assay was utilized to evaluate initial apoptosis induction in all cells and In-cell western assay was used to detect protein expression levels associated with mitochondrial apoptosis, cell cycle, EGFR and its downstream AKT and ERK1/2 pathways. Molecular docking studies revealed that most estrone analogs exhibited improved potency and binding compared to the positive controls, erlotinib and sorafenib when docked against the EGFR kinase-domain (pdb codes: 2ITW, 1M17). Subsequently, several estrone analogs exhibited significant cytotoxic effects against the different cancer cell lines in vitro. Notably, MMA307 and MM320 recorded lower IC50 molarities of 2.88 ± 0.21 and 9.68 ± 0.24 μM compared to the positive control, sorafenib, IC50 value of 20.62 ± 1.32 μM in NCIH226 cells. Similarly, administration of MMA307 and MMA321 to MDA-MB-468 cells yielded potent IC50 concentrations of 0.85 ± 0.00 and 0.56 μM ± 0.01 μM, respectively, when compared to sorafenib, IC50 value of 10.09 ± 0.68 μM. Furthermore, Fz25 recorded IC50 dose of 8.13 ± 0.15 μM in MDA-MB-231 cell lines when compared to sorafenib, 12.21 ± 0.96 μM. Treatment with the MMA307 and MMA320 resulted in downregulation of Dyrk1B (dual-specificity tyrosine phosphorylation-regulated kinase B), cyclin D1 and concomitant upregulation of phospho-cyclin D1 and p21waf1/cip1 contributing to cell cycle arrest in the G1 phase. Also, downregulation of EGFR and phospho-EGFR levels as well as suppression of activated MAP kinase signaling proteins-phospho-B-Raf, phospho-MEK1/2 and phospho-ERK1/2 were observed after NCIH226 cells were exposed to estrone analogs. Similarly, MMA307 and MMA321 downregulated cyclin D1 expression levels resulting in G1 phase cell cycle arrest in MDA-MB-468 cells. Also, these compounds halted the MAPK and AKT signaling pathways due to their ability to downregulate EGFR and activated EGFR expressions. Moreover, mitochondrial apoptosis was induced in the TNBC model, MDA-MB-468, upon MMA307 and MMA321 exposures. In a different study, treatment of MDA-MB-231 cells with Fz25 induced mitochondrial apoptosis and contributed to the G1 phase of cell cycle arrest due to decreased expressions of cyclin D1 and Dyrk1B. Interestingly, this compound impacted the MAPK and AKT signaling pathways due to its ability to decrease EGFR and activated EGFR expressions. To conclude, the present study is the first to report on the cytotoxic potential of novel estrone analogs and provide evidence that MMA307, MMA320, MMA321 and Fz25 are promising novel lead compounds. Further investigations are needed to develop these potent compounds as the next generation anti-EGFR therapies for treating serious EGFR dependent lung and breast cancers.

Library of Congress Subject Headings

Lungs -- Cancer -- Treatment.
Breast -- Cancer -- Treatment.
Epidermal growth factor -- Receptors.



Number of Pages



South Dakota State University



Rights Statement

In Copyright